A major component of barrier function in stratified squamous epithelia is the cornified cell envelope (CE). This is a multi-component 15 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid envelope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the crosslinking of several structural proteins by transglutaminases. Studies on the biology and assembly of the protein and lipid components are a major effort of this laboratory. Specifically, we are studying: (i) the crosslinking of proteins in CEs isolated from a variety of sources to explore which proteins are crosslinked together through which glutamines and lysines, and to provide information on structure and function; (ii) two key structural proteins, loricrin and the small proline rich protein (SPR) families; and (iii) the ceramide lipids which become covalently attached to the CE. CEs We are studying the features of CEs isolated from human foreskin epidermal stratum corneum, immature terminally differentiating foreskin epidermis, cultured human epidermal keratinocytes induced to terminally differentiate, mouse forestomach epithelium, and human gingiva epithelium. We have used controlled proteolysis to dissect apart the CEs, separate crosslinked peptides, and perform protein sequencing. In all cases except the gingiva, the bulk of the protein envelope consists of loricrin (70-80%) admixed with smaller amounts (2-20%) of SPRs. The gingiva CEs contain <50% SPRs. In all cases, the SPRs appear to function as promiscuous crossbridging proteins, by linking together various proteins, most often loricrin or themselves, through multiple adjacent glutamine and lysine residues on their head and tail domains only. In addition, we have found that there is a direct correlation between the amount of SPRs present in CEs and the presumed physical characteristics and exposure to physical trauma of the epithelium: human or mouse trunk CEs contain little or no SPRs; human foreskin epidermal CEs 5% SPRs; mouse foot pad and lip epidermal CEs 10% SPRs; mouse forestomach CEs 20% SPRs; and gingiva CEs <50% SPRs. These data suggest that crossbridging SPRs serve to modulate the biomechanical properties of the CEs in which they are expressed. Moreover, we have shown that the CE is crosslinked to the keratin intermediate filament cytoskeleton, which further suggests that the SPRs may contribute in important ways to the biomechanical properties and requirements of an entire epithelium. In addition, we have recovered many crosslinks involving the keratins, involucrin, and other cell junctional proteins including desmoplakin and envoplakin. Our data are consistent with the possibility that CE assembly may be initiated at the cell periphery near the site of where keratin filaments meet desmosomes. Further sequencing on these different types of CEs is in progress. Loricrin We have expressed human loricrin in bacteria and used it to characterize its structure, biochemical properties, and crosslinking by epidermal transglutaminases (TGase) in vitro. By biophysical measurements it has some structure in solution associated with its multiple tyrosines. It is a complete TGase substrate because it is oligomerized by all three epidermal TGases in in vitro reactions, although with different kinetic efficiencies, and utilization of different glutamines and lysines. From comparisons of the residues used in vitro with those used in vivo from sequencing of CEs, we can conclude that both TGase 1 and TGase 3 are required for the correct crosslinking of loricrin in vivo. Future studies will be aimed at determining the structure of loricrin by use of solution nmr methods on either full length expressed protein, or synthetic peptides from selected portions of it. The proximal promoter of the human loricrin gene resides within the first 160 bp above the transcription initiation start site. Interactions of c-fos/c-jun proteins at an AP1 site are essential for epithelial expression in keratinocytes. Unlike the mouse loricrin gene, a calcium responsive element lies within this region. We are actively characterizing a series of negative elements which lie just upstream of the 160 bp region. Small proline rich proteins SPRs consist of three distinct families consisting of from one to 11 members. We have expressed one member of each of the human SPR1, SPR2 and SPR3 proteins for in vitro studies. By circular dichroism, they have little organized structure in solution. What structure is present can be attributed to the central proline-rich peptide repeats, and the signal strength is proportional to the number of repeats. The SPR proteins are also complete substrates in in vitro crosslinking reactions for the three TGases commonly expressed in the epidermis. In all cases of SPR proteins studied, the glutamines and lysines used for crosslinking are located only on the end domains, suggesting they may function as crossbridging proteins. However, the details are different. In the case of SPR2 proteins, the TGase 1 enzyme uses only one glutamine residue on the head domain, and only one lysine on the tail domain for interchain crosslinking, whereas the TGase 3 enzyme uses multiple head and tail residues for interchain crosslinking. In the SPR1 proteins, we found that there are two head domain regions termed head A and head B. The former are used only by the TGase 3 enzyme for interchain crosslinking, whereas the latter are used only by the TGase 1 enzyme primarily for intrachain crosslinking. A similar situation was found for SPR3 proteins. Moreover, we correlated these crosslinking data with those for loricrin, and we found that the TGase 1 and 3 enzymes crosslink at common sites on the respective proteins. These data suggest that TGase 3 initiates crosslinking of loricrin and SPRs into small interchain oligomers which are later crosslinked to the CE by the TGase 1 enzyme. Solution nmr structural studies on the SPR2 and 3 proteins are in progress. Preliminary data suggest the central peptide repeat domains adopt novel omega-loop-like protein folds.